The Kuhn-Roth Procedure

1 The Kuhn-Roth Procedure (Kuhn and Roth 1933, Roth 1953) 

Methanol, refluxed over solid potassium hydroxide (pellet form) for 15min to remove residual acids and distilled, b.p. 64-65 °C 

Sodium hydroxide solution, 1M in methanol-water dissolve 4g of sodium hydroxide (pellet form) in 50ml of water (Note 2) and 50ml of methanol 

Sulfuric acid, 6.5 M add 100 ml of concentrated sulfuric acid to 200ml of cold water (Note 2) 

Phenolphthalein solution, 1% dissolve lg of phenolphthalein in a mixture of 90 ml of ethanol and 10 ml of water 

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Reduction apparently creates fresh hydroaromatic structure (at the expense of the aromatics), and thus the methyl groups attached to the aromatic structures are likely to become amenable to quantitative estimation (by the Kuhn-Roth procedure), provided that the particular aromatic ring is reduced to hydroaromatic. Significantly, the reduced samples of the lower rank coals did not yield much higher values for methyl groups than the original samples. Thus, it would appear that Kuhn-Roth estimation does not completely measure the true C-methyl content in coals, especially in high rank coal samples. 

Functional Group Analysis. The total hydroxyl content of lignin is determined by acetylation with an acetic anhydride—pyridine reagent followed by saponification of the acetate, and followed by titration of the resulting acetic acid with a standard 0.05 W sodium hydroxide solution. Either the Kuhn-Roth (35) or the modified Bethge-Liadstrom (36) procedure may be used to determine the total hydroxyl content. The aUphatic hydroxyl content is determined by the difference between the total and phenoHc hydroxyl contents. 

In 1947, Kinney (15) drew attention to a direct relationship between the amount of acetic acid formed on oxidizing coal with nitric acid and potassium dichromate and the yields of methane obtained by low temperature carbonization. The oxidation method leading to acetic acid is a variation of the standard Kuhn-Roth procedure, and hence what Kinney indicated as the methane yielding structure is obviously the methyl groups in coal. However, the relationship he pointed out can only be qualitative since true C-methyl content (as shown in the present work) cannot be obtained by any... 

Martin B. Neuworth The Kuhn-Roth method has a number of limitations. These were discussed by Brandenberger et al. (Anal. Chem. 33, 453 (1961)). The normal method for estimating C-methyl groups is based on a determination of total acidity calculated as acetic acid. Brandenberger has shown that other acids can be produced during the oxidation—e.g., benzoic acid, and these acids steam distil with the acetic acid. Therefore, when the classical Kuhn-Roth procedure is applied to a substance like coal, it would lead to methyl group values of questionable significance. 

In experiment 1 (Table VII), the precursors emodin (76) and emodin-anthrone (77) were labeled with the same isotope ( " C) in two different positions [at C(3) and C(ll)] by the synthetic procedures described above. After incorporation, the corresponding carbon atoms of the ergochrome EE (2) could be analyzed as acetic acid by Kuhn-Roth oxidation. The specific incorporations were derived from the activities of the two carbon atoms, determined separately by Schmidt degradation of the acetic acid. The results show that emodinanthrone (77) is incorporated into ergochrome EE (2) 4.5 times more effectively than emodin (76). 

Hydrolysis of the labeled compound with 2 N sulfuric acid furnished 3-amino-4-methyl-2-hexanone which, on treatment with iodine in the presence of sodium hydroxide, furnished isoleucine and iodoform. The iodoform was oxidized to carbon dioxide, while treatment of isoleucine with ninhydrin furnished 2-methylbutanal, isolated as its 2,4-dinitrophenylhydrazone. Kuhn-Roth oxidation of the aldehyde gave acetic acid which was degraded by the Schmidt procedure to carbon dioxide and methylamine. The 2-methylbutanal was also degraded stepwise by the method of Strassman. Determination of the radioactivity of the various degradation products showed that 94% of the activity was equally shared between the lactam C(2) and side-chain C(10) carbonyl atoms. The remaining activity was shared between C(4) of the lactam ring and the a> carbon atom of the ec-butyl side chain. The results are consistent with derivation of tenuazonic acid from isoleucine and two molecules of acetic acid. However, the direct incorporation of isoleucine into tenuazonic acid was not investigated. 

From these results it was deduced that any radioactivity in the acetic acid obtained by Kuhn-Roth oxidation of [2-acetate-labeled erythroskyrin must be derived from C(26). The acetic acid contained 4.45% of the total activity, and this was shown by the Schmidt degradation procedure to be located in the methyl group. The acetic acid obtained by Kuhn-Roth oxidation of [1- " C]- and [2- C]malonate-derived erythroskyrin was inactive. These results proved that the biosynthesis of erythroskyrin involved the condensation of valine and a polyketide moiety. More specifically, the results were taken as evidence that erythroskyrin is biosynthesized from valine [N(l) and C(4) to C(8)], one acetate [C(25) and C(26)], and nine malonate [C(2), C(3), C(9) to C(24)] units. This conclusion must, however, be considered as tentative, particularly as no direct evidence was obtained on the incorporation of from the various substrates into C(2) and... 

Kuhn-Roth oxidation of O-methyldeacetylcyclopiazonic acid (14) yielded 0.34 mole of acetic acid, which was degraded by the Schmidt procedure to carbon dioxide, and methylamine, isolated as A-methylnitroaniline. The A-methylnitroaniline obtained in this way accounted for 96% of the total activity, while the carbon dioxide from acetate-derived aCA con-... 

Kuhn and Roth developed a procedure in which hydrolysis and subsequent distillation of acetic acid were carried out in a single flask. The main objection appears to be that the design of the apparatus permits the carryover of sulphuric acid, either in mist or droplet form if rapid distillation or bumping occurs. 

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